EP2261336B1 - DNA-Sequenz und rekombinante Herstellung von Gruppe-4 Majorallergenen aus Getreiden - Google Patents
DNA-Sequenz und rekombinante Herstellung von Gruppe-4 Majorallergenen aus Getreiden Download PDFInfo
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- EP2261336B1 EP2261336B1 EP10009677.5A EP10009677A EP2261336B1 EP 2261336 B1 EP2261336 B1 EP 2261336B1 EP 10009677 A EP10009677 A EP 10009677A EP 2261336 B1 EP2261336 B1 EP 2261336B1
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- dna
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- allergens
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to the provision of DNA sequences of group-4 major allergens from cereals ( Triticeae ).
- the invention also includes fragments, new combinations of partial sequences and point mutants with hypoallergenic activity.
- the recombinant DNA molecules and the deduced polypeptides, fragments, recombinations of partial sequences and variants can be used for the therapy of pollen allergic diseases.
- the recombinantly produced proteins can be used for in vitro and in vivo diagnostics of pollen allergies.
- Allergies of type 1 have worldwide significance. Up to 20% of the population in industrialized countries suffer from conditions such as allergic rhinitis, conjunctivitis or bronchial asthma. These allergies are caused by airborne allergens (aeroallergens) released from sources of various origins such as plant pollen, mites, cats or dogs. Up to 40% of these type 1 allergy sufferers in turn show specific IgE reactivity with grass pollen allergens, including cereal pollen allergens ( Freidhoff et al., 1986, J. Allergy Clin. Immunol. 78, 1190-2001 ). Of particular importance among the pollen allergens are the allergens of rye.
- the type 1 allergy-causing substances are proteins, glycoproteins or polypeptides. These allergens react after absorption via the mucous membranes with the sensitized persons at the Surface of mast cell-bound IgE molecules. If two IgE molecules are cross-linked by an allergen, this leads to the release of mediators (eg histamine, prostaglandins) and cytokines by the effector cell and thus to the corresponding clinical symptoms.
- mediators eg histamine, prostaglandins
- the allergens from the pollen of different species of the grass family are divided into groups that are homologous with each other.
- the molecules of major allergen group 4 have a high immunological cross-reactivity with both mouse monoclonal antibodies and with human IgE antibodies ( Fahlbusch et al., 1993 Clin. Exp. Allergy 23: 51-60 ; Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98: 1065-1072 ; Su et al., 1996, J. Allergy Clin. Immunol. 97: 210 ; Fahlbusch et al., 1998, Clin. Exp. Allergy 28: 799-807 ; Gavrovic-Jankulovic et al., 2000, Invest. Allergol. Clin. Immunol.
- the object underlying the present invention was therefore to provide DNA sequences of group-4 major allergens from cereals, in particular the allergen Sec c 4 from rye ( Secale cerale ) (SEQ ID NO 1, 3), Hor v 4 from barley ( Hordeum vulgare ) (SEQ ID NO 5) and Tri a 4 from wheat ( Triticum aestivum ) (SEQ ID NO 7, 9) as well as corresponding recombinant DNA molecules, based on which the allergens expressed as protein and a pharmacologically significant utilization as such or in a modified form can be made accessible.
- the sequence of Phl p 4 (SEQ ID NO 11) was the starting point for the present invention.
- the present invention therefore relates to a DNA molecule having the nucleotide sequence according to SEQ ID NO 5.
- the invention further relates to the DNA sequences according to the invention homologous sequences or corresponding DNA molecules of group 4 allergens from other Poaceae such as Lolium perenne, Dactylis glomerata, Poa pratensis, Cynodon dactylon and Holcus lanatus, due to the existing sequence homology with hybridize the DNA sequences according to the invention under stringent conditions, or have an immunological cross-reactivity with respect to the allergens according to the invention.
- Poaceae such as Lolium perenne, Dactylis glomerata, Poa pratensis, Cynodon dactylon and Holcus lanatus
- the invention also includes fragments, new combinations of partial sequences and point mutants with hypoallergenic activity.
- the invention therefore furthermore relates to corresponding partial sequences, a combination of partial sequences or exchange, elimination or addition mutants which code for an immunomodulatory, T cell-reactive fragment of a Poaceae group 4 allergen.
- the present invention therefore also provides a DNA molecule described above or below or a corresponding recombinant expression vector as a pharmaceutical.
- the corresponding recombinantly produced proteins can be used for therapy as well as for in vitro and in vivo diagnostics of pollen allergies.
- the cloned nucleic acid is ligated into an expression vector and this construct is expressed in a suitable host organism. After biochemical purification, this recombinant allergen is available for the detection of IgE antibodies in established procedures.
- the present invention therefore furthermore relates to a recombinant expression vector comprising a DNA molecule described above or below, functionally linked to an expression control sequence and a host organism, transformed with said DNA molecule or said expression vector.
- Also contemplated by the invention is the use of at least one previously described DNA molecule or at least one expression vector described above for the manufacture of a medicament for immunotherapeutic DNA vaccination of patients with allergies, at their release Group 4 allergens of Poaceae, preferably Triticeae , especially Sec c , Hor v 4, Tri a 4, are involved and / or for the prevention of such allergies.
- the invention can be used as an essential component in a recombinant allergen- or nucleic acid-containing preparation for specific immunotherapy.
- the unchanged protein in the primary structure can be part of the preparation.
- the nucleic acid per se when ligated with a eukaryotic expression vector, creates a preparation which directly adjusts the allergic immune state in the therapeutic sense.
- the present invention is the polypeptides encoded by one or more of the previously described DNA molecules, preferably in their capacity as a drug. It is the protein corresponding to an amino acid sequence according to SEQ ID NO 6. In particular, it is the mature protein (without signal sequence portion), starting with the amino acid 23 (SEQ ID NO 6). Furthermore, the invention relates to proteins which contain these amino acid sequences or parts of these sequences,
- the invention accordingly also relates to a process for producing such polypeptides by cultivating a host organism and recovering the corresponding polypeptide from the culture.
- Also contemplated by the invention is the use of at least one polypeptide described above for the manufacture of a medicament for the diagnosis and / or treatment of allergies, at their triggering group 4 allergens of Poaceae , preferably Triticeae, in particular
- homologous EST fragments could be found in EST databases of Hordeum vulgare . However, these fragments do not overlap to a complete gene. However, based on the EST sequences found, Hor v 4 specific primers could be generated which were used for amplification of the Hor v 4 gene from genomic DNA.
- a total of 15 clones were analyzed. 4 clones were obtained by PCR with primers # 195 and # 198. 4 clones were obtained by PCR with primers # 195 and # 202. 3 clones were obtained by PCR with primers # 194 and # 198. 4 clones were obtained by PCR with primers # 194 and # 202.
- the deduced protein sequence begins within the signal sequence of Hor v 4 and extends to the C-terminal end of the protein (from amino acid 23 of SEQ ID NO 6).
- a total of 13 clones were analyzed. 4 clones were obtained by PCR with primers # 204 and # 203. 4 clones were obtained by PCR with primers # 204 and # 199. 3 clones were obtained by PCR with primers # 206 and # 203. 4 clones were obtained by PCR with primers # 206 and # 199.
- the deduced protein sequences begin within the signal sequence of tri a 4 and extend to the C-terminal end of the protein. Two variants Tri a 4.01 (from amino acid 22 of SEQ ID NO 8) and Tri a 4.02 (from amino acid 22 of SEQ ID NO 10) were found.
- the DNA sequences according to SEQ ID NO 1, 3, 5, 7 and 9 were incorporated into expression vectors (eg pProEx, pSE 380).
- expression vectors eg pProEx, pSE 380.
- E. coli optimized codons used for the N-terminal amino acids known from protein sequencing.
- This diagnosis can be made in vitro by detection of specific antibodies (IgE, IgG1-4, IgA) and reaction with IgE-loaded effector cells (eg basophils from the blood) or in vivo by skin test reactions and provocation on the reaction organ ,
- the reaction of the allergens according to the invention with T-lymphocytes of grass pollen allergy sufferers can be achieved by the allergen-specific stimulation of the T lymphocytes for proliferation and cytokine synthesis both with T cells in freshly prepared blood lymphocytes and on established nSec c 4, nHor v 4 and nTri a 4, respectively.
- reactive T-cell lines and clones can be detected.
- Site-directed mutagenesis has altered the cysteine-encoding triplets to encode other amino acids, preferably serine. Variants were prepared in which single cysteines were exchanged, as well as those in which different combinations of 2 cysteine residues or all cysteines were changed. The expressed proteins of these cysteine point mutants show greatly reduced or absent reactivity with IgE antibodies of allergy sufferers, but react with the T lymphocytes of these patients.
- the present invention therefore furthermore relates to a DNA molecule described above or below in which one or more or all of the cysteine residues of the corresponding polypeptide have been exchanged for another amino acid by site-directed mutagenesis.
- hypoallergenic fragments corresponding to polypeptides with T-cell epitopes as well as those of the hypoallergenic point mutants can be detected by their reaction with T-cells of grass pollen allergy.
- Such hypoallergenic fragments or point mutants of the cysteines can be used as preparations for the hyposensitization of allergy sufferers since they react with equal effectiveness with the T cells, but due to the diminished or completely absent IgE reactivity lead to lower IgE-mediated side effects.
- nucleic acids coding for the hypoallergenic allergen variants according to the invention or the unchanged DNA molecules according to the invention are ligated with a human expression vector, these constructs can also be used as preparations for immunotherapy (DNA vaccination).
- the present invention relates to pharmaceutical preparations containing at least one previously described DNA molecule or at least one expression vector described above and optionally other active ingredients and / or adjuvants for immunotherapeutic DNA vaccination of patients with allergies at their release group 4 allergens the Poaceae , preferably Triticeae , especially Sec c 4, Hor v 4, Tri a 4, are involved and / or for the prevention of such allergies.
- Another group of pharmaceutical preparations according to the invention contains at least one previously described polypeptide instead of the DNA and is suitable for the diagnosis and / or treatment of said allergies.
- compositions according to the present invention containing as active ingredients a polypeptide according to the invention or an expression vector and / or their respective pharmaceutically usable derivatives, including mixtures thereof in all ratios.
- the active compounds according to the invention can be brought into a suitable dosage form together with at least one solid, liquid and / or semi-liquid carrier or excipient and optionally in combination with one or more further active ingredients.
- excipients immunostimulatory DNA or oligonucleotides with CpG motifs are particularly suitable.
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pulmonology (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PL10009677T PL2261336T3 (pl) | 2003-12-16 | 2004-12-01 | Sekwencja dna i rekombinacyjne otrzymywanie alergenów głównych grupy 4 ze zbóż |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10359351A DE10359351A1 (de) | 2003-12-16 | 2003-12-16 | DNA-Sequenz und rekombinante Herstellung von Gruppe-4 Majorallergenen aus Getreiden |
| EP04803421A EP1709177B1 (de) | 2003-12-16 | 2004-12-01 | Dna-sequenz und rekombinante herstellung von gruppe-4 majorallergenen aus getreiden |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP04803421.9 Division | 2004-12-01 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| EP2261336A1 EP2261336A1 (de) | 2010-12-15 |
| EP2261336B1 true EP2261336B1 (de) | 2013-10-16 |
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Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP10009677.5A Expired - Lifetime EP2261336B1 (de) | 2003-12-16 | 2004-12-01 | DNA-Sequenz und rekombinante Herstellung von Gruppe-4 Majorallergenen aus Getreiden |
| EP04803421A Expired - Lifetime EP1709177B1 (de) | 2003-12-16 | 2004-12-01 | Dna-sequenz und rekombinante herstellung von gruppe-4 majorallergenen aus getreiden |
| EP10009678.3A Expired - Lifetime EP2261337B1 (de) | 2003-12-16 | 2004-12-01 | DNA-Sequenz und rekombinante Herstellung von Gruppe-4 Majorallergenen aus Getreiden |
Family Applications After (2)
| Application Number | Title | Priority Date | Filing Date |
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| EP04803421A Expired - Lifetime EP1709177B1 (de) | 2003-12-16 | 2004-12-01 | Dna-sequenz und rekombinante herstellung von gruppe-4 majorallergenen aus getreiden |
| EP10009678.3A Expired - Lifetime EP2261337B1 (de) | 2003-12-16 | 2004-12-01 | DNA-Sequenz und rekombinante Herstellung von Gruppe-4 Majorallergenen aus Getreiden |
Country Status (14)
| Country | Link |
|---|---|
| US (3) | US7935347B2 (cg-RX-API-DMAC7.html) |
| EP (3) | EP2261336B1 (cg-RX-API-DMAC7.html) |
| JP (1) | JP4960102B2 (cg-RX-API-DMAC7.html) |
| CN (2) | CN103305531A (cg-RX-API-DMAC7.html) |
| AT (1) | ATE501257T1 (cg-RX-API-DMAC7.html) |
| AU (1) | AU2004299600A1 (cg-RX-API-DMAC7.html) |
| BR (1) | BRPI0417736A (cg-RX-API-DMAC7.html) |
| CA (3) | CA2549573C (cg-RX-API-DMAC7.html) |
| DE (2) | DE10359351A1 (cg-RX-API-DMAC7.html) |
| ES (3) | ES2363015T3 (cg-RX-API-DMAC7.html) |
| PL (3) | PL2261337T3 (cg-RX-API-DMAC7.html) |
| PT (3) | PT2261336E (cg-RX-API-DMAC7.html) |
| RU (1) | RU2373283C2 (cg-RX-API-DMAC7.html) |
| WO (1) | WO2005059136A2 (cg-RX-API-DMAC7.html) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10359351A1 (de) * | 2003-12-16 | 2005-07-21 | Merck Patent Gmbh | DNA-Sequenz und rekombinante Herstellung von Gruppe-4 Majorallergenen aus Getreiden |
| US9920101B2 (en) | 2014-03-31 | 2018-03-20 | Boehringer Ingelheim Vetmedica Gmbh | Modular antigen transportation molecules and uses therof |
| KR20180061338A (ko) | 2015-09-30 | 2018-06-07 | 베링거잉겔하임베트메디카게엠베하 | 동물에서의 개선된 모듈러 항원 수송 분자 및 이의 용도 |
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| US5762943A (en) * | 1996-05-14 | 1998-06-09 | Ribi Immunochem Research, Inc. | Methods of treating type I hypersensitivity using monophosphoryl lipid A |
| DE19918682A1 (de) * | 1999-04-23 | 2000-10-26 | Merck Patent Gmbh | DNA-Sequenz und rekombinante Herstellung eines Graminaen-Allergens |
| US7214786B2 (en) * | 2000-12-14 | 2007-05-08 | Kovalic David K | Nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement |
| EP1402037A1 (en) * | 2001-06-22 | 2004-03-31 | Syngenta Participations AG | Plant genes involved in defense against pathogens |
| AUPR779201A0 (en) * | 2001-09-20 | 2001-10-11 | University Of Melbourne, The | Immunotherapeutic and immunoprophylactic reagents |
| WO2003025008A2 (en) | 2001-09-21 | 2003-03-27 | Affinium Pharmaceuticals, Inc. | Bacterial polypeptides involved in protein processing |
| CA2491038C (en) * | 2002-06-25 | 2017-04-25 | Merck Patent Gesellschaft Mit Beschraenkter Haftung | Dna sequence and preparation of grass pollen allergen phl p4 by recombinant methods |
| DE10359351A1 (de) * | 2003-12-16 | 2005-07-21 | Merck Patent Gmbh | DNA-Sequenz und rekombinante Herstellung von Gruppe-4 Majorallergenen aus Getreiden |
-
2003
- 2003-12-16 DE DE10359351A patent/DE10359351A1/de not_active Withdrawn
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2004
- 2004-12-01 DE DE502004012294T patent/DE502004012294D1/de not_active Expired - Lifetime
- 2004-12-01 PT PT100096775T patent/PT2261336E/pt unknown
- 2004-12-01 EP EP10009677.5A patent/EP2261336B1/de not_active Expired - Lifetime
- 2004-12-01 CN CN2013102182194A patent/CN103305531A/zh active Pending
- 2004-12-01 CA CA2549573A patent/CA2549573C/en not_active Expired - Fee Related
- 2004-12-01 PL PL10009678T patent/PL2261337T3/pl unknown
- 2004-12-01 ES ES04803421T patent/ES2363015T3/es not_active Expired - Lifetime
- 2004-12-01 JP JP2006544263A patent/JP4960102B2/ja not_active Expired - Fee Related
- 2004-12-01 PT PT04803421T patent/PT1709177E/pt unknown
- 2004-12-01 CA CA2945267A patent/CA2945267A1/en not_active Abandoned
- 2004-12-01 CA CA2873261A patent/CA2873261C/en not_active Expired - Fee Related
- 2004-12-01 EP EP04803421A patent/EP1709177B1/de not_active Expired - Lifetime
- 2004-12-01 RU RU2006125511/13A patent/RU2373283C2/ru not_active IP Right Cessation
- 2004-12-01 AU AU2004299600A patent/AU2004299600A1/en not_active Abandoned
- 2004-12-01 ES ES10009678.3T patent/ES2443302T3/es not_active Expired - Lifetime
- 2004-12-01 US US10/583,089 patent/US7935347B2/en not_active Expired - Fee Related
- 2004-12-01 ES ES10009677.5T patent/ES2442951T3/es not_active Expired - Lifetime
- 2004-12-01 PL PL04803421T patent/PL1709177T3/pl unknown
- 2004-12-01 BR BRPI0417736-3A patent/BRPI0417736A/pt not_active Application Discontinuation
- 2004-12-01 PT PT100096783T patent/PT2261337E/pt unknown
- 2004-12-01 CN CNA2004800372314A patent/CN1894410A/zh active Pending
- 2004-12-01 EP EP10009678.3A patent/EP2261337B1/de not_active Expired - Lifetime
- 2004-12-01 PL PL10009677T patent/PL2261336T3/pl unknown
- 2004-12-01 WO PCT/EP2004/013664 patent/WO2005059136A2/de not_active Ceased
- 2004-12-01 AT AT04803421T patent/ATE501257T1/de active
-
2010
- 2010-11-16 US US12/947,361 patent/US9309297B2/en not_active Expired - Fee Related
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2015
- 2015-12-17 US US14/972,633 patent/US20170246317A1/en not_active Abandoned
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